The background description includes information that may be useful in understanding the methods described herein. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed subject matter, or that any publication specifically or implicitly referenced is prior art.
The advent of molecular biology combined with various immunological techniques has led to the development of numerous therapeutic biologic agents, including monoclonal antibodies, fusion proteins, recombinant receptor antagonists, and more recently, CAR T-cell therapies. With the ongoing expansion of T cell based therapeutic approaches, techniques to isolate activated, functional T cells with high purity are needed in order to conduct various T cells assays, including activation, differentiation, expansion, and signaling assays.
Various cell sorting technologies exist for concentrating and isolating cells, including centrifugation, membrane filtration, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), microfluidics, etc. Some of these techniques isolate or concentrate cells in bulk, e.g., such as centrifugation and filtration, while other techniques (e.g., FACS, MACS) allow higher selectivity.
Flow cytometry allows randomly distributed cells to be focused into a single continuous flow stream. Once in this continuous stream, each cell travels at an approximately identical speed, and passes through an optical detection zone for analysis based upon its optical properties (e.g., scatter and autofluorescence). Each cell passing through the optical detection zone may receive the same illumination intensity and time for reliable and consistent optical detection and analysis (see, e.g., Cram, Meth. Cell. Sci. (2002) 24(1): 1-9). In some cases, a modified type of flow cytometer, referred to as a fluorescence-activated cell sorter (FACS) may be employed. This type of device includes a sorting capability downstream of the optical detection zone, to select cells (e.g., expressing a fluorescent marker, labeled with a fluorescent tag, etc.) detected upstream.
Other approaches include using dielectrophoresis (DEP) for particle sorting. For example, a 3D, tunable, sheathless cell focusing and sorting DEP-based device has been used to separate particles based on size (see, Y. C. Kung et al., Small (2016) 12:4343; U.S. application Ser. No.: 15/484,964; U.S. Publication No.: 2015/0041325).
Despite the various techniques that are available for isolating cells, many of these techniques rely on modification of the cell, e.g., to express a fluorescent label, to be surface-labeled with a fluorescent marker, etc.), which is generally not desirable for therapeutic grade T cell production. Further, it is difficult to isolate activated T cells, e.g., from a non-activated T cells, in a manner that leads to high purity.
All publications identified herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments described herein may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
Unless the context dictates the contrary, all ranges set forth herein should be interpreted as being inclusive of their endpoints and open-ended ranges should be interpreted to include only commercially practical values. Similarly, all lists of values should be considered as inclusive of intermediate values unless the context indicates the contrary.
As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the subject matter described herein and claimed below and does not pose a limitation on the scope of the subject matter otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the claimed subject matter.
Groupings of alternative elements or embodiments of the subject matter disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.